Process for the manufacture of vaccines



Patented Aug. 29, 1933 PROCESS FOR THE MANUFACTURE OF VACCINES WernerWeichlein, Prenzlau, Germany No Drawing. Application December 20, 1927,Serial No. 241,455, and in Germany December 2 Claims.

It is well-known that men and animals are protected from or cured ofinfectious diseases by means of inoculation. Living, enfeebled or deadmorbific agents (germs) or infectious matter 5 or its products are usedas inoculation vaccines which are obtained by various different ways andmethods, while the morbific agents are cultivated by artificial means ortheir virulence is modified by being passed through suitable animals.

It is also already known how to kill or render uninjurious thesemorbific agents or their products by aniline dyes or aniline, or how toavoid infectious diseases by introducing pigments in the bodies ofanimals. In this way it has not, however, been possible to producevaccines which can be given in exact doses and are immediatelyapplicable without causing complication.

The object of the invention is a process for the manufacture ofinoculation vaccines, whereby the infectious matter (virus) or themorbific agents or their products are mixed with weak solutions ofaniline dyes and the mixture kept at a temperature of blood heat (37 C.)for one to two days. By the term infectious matter I mean the germs inpure culture or in mixture, and they may be contained in liquids, etc.Thus the pathogenic element is broken up, although the immunifyingeffect is retained. Various clearly defined solutions of aniline dyes,which are used in certain quantities, are suitable for the separatemorbific agents. Thus a 5% methyl blue solution is especially suited tothe treatment of red murrain bacillus, for the bacteria paratyphyenteritidis (Giirtner), which gives rise to poisoning in human beings, abrilliant green solution is most suitable, and a 5% fuchsine solutionfor the birds cholera bacillus, and so forth.

The morbific agents or infectious matter can, in accordance with thisinvention, be so influtransferred to artificial soil and test animalscan no longer contract disease through them, or

the effect may be that, on being transferred to artificial soil, 8.growth and an increase take place as in the case of an ordinary culture,although test animals can no longer contract diseases. The virulence ofthe morbific agents or of the infectious matter can thus be eliminatedabsolutely at will. A subsequent alteration of the inoculation vaccinedoes not appear. The latter therefore remains constant, which, in viewof the intended effect, is exceedingly important. According'to theinvention, the manufacture of effectual inoculation vaccines is thussuccessfully undertaken if bacteria cultures, and espepossible.

enced that either they no longer grow after being cially morbific agentsof animals or plant-type, which either themselves or through theirpoisons contain liquids-many morbific agents (germs) have not, however,been isolated as yet, such as those of the foot and mouth disease andsmallpox-are kept, after the addition of pigments, at a blood-heattemperature for 24 to 48 hours.

According to this invention it is possible to deprive the infectiousmatter (virus) which develops those diseases, the germs of which are notknown, of its infectious qualities and to use said infectious matterafter being treated with aniline dyes as inoculation for man and animalsto protect them against infection.

Such substances are for instance the extracts of organs of animalssuffering of such diseases, or liquids or body fluids containing theinfectious matter just referred to.

Up to the present, the medical faculty has not been successful inproducing a highly immunifying serum by transferring through inoculationenfeebled or dead morbific agents and infectious matter to serumanimals. For this purpose, living morbific agents had to be used to beefiective. With the new inoculation vaccine alone, howso ever, it ispossible to produce a serum, even in the case of red murrain disease,containing over 200 units of immunity, which has hitherto been im- Thisfact is very important for the reason that the transfer of livingmorbific agents 851 to serum animals by inoculation, in fever caseswhich are frequently of long duration, causes inflammati'on of thejoints and the setting in of poisoning. By the use of the newinoculation vaccine, on the other hand, these complications, which veryoften cause the death of the serum animal, do not occur.

The process is carried out in the following manner: 4

Watery solutions of aniline dyes, such as fuchsine or brilliant green,are mixed with watery deposits of bacterial or infectious matter (e. g.in physiological salt solution). These mixtures have then to be shakenthoroughly and to be kept at a temperature of 37 C. for 24 hours orlonger, as only at a temperature of blood-heat do the bacteria orinfectious germs and the aniline dyes. react or become incorporated.

In the place of watery deposits of bacteria can be used bouilloncultures or fluids which contain bacteria or infectious matter.

A detailed description of the production of one vaccine, i. e., apigmentary vaccine against the infectious calving (abortus bang) isherein given:Sterile nutrient broth is inoculated in retorts eachcontaining 2 litres by pouring liquid Abortus Bang cultivation over itand depositing it in an incubator of 37 C. to promote the growth of thebacteria. In it the Abortus Bang bacteria will grow luxuriantly within10-12 days. This cultivation is then microscopically examined in orderto ascertain whether the cultivation has remained pure, i. e. whetheronly Abortus Bang bacilla have been growing in same. This being thecase, 300 cc. of a 2% aqueous methylene-blue solution is admixed toevery retort which, as stated above, has a capacity of 2 litres. Afterthis the retorts are stirred thoroughly and placed into an incubator of37 C. for a time of 48 hours. By the addition of the dye and thesimultaneous heating up to body-temperature the Abortus Bang bacteriaare thus killed. After 48 hours have passed, the prepared vaccine iscarefully examined to ensure that all of the bacteria have been entirelykilled by the preceding procedure. This is effected by a small quantityof the vaccine being deposited upon one solid and one liquid nutrientmedium each, and placing the latter once more into the incubator, with aview to seeing whether a further growth of the bacteria will result.Should this not be the case in the little test-tube, the vaccine ispoured into the ampoules most appropriate for practical treatment andthen dispatched ready for sale. This is of course only an example, butthe process is essentially the same in producing vaccines against otherdiseases, although the bacteria and particular aniline dyemay bechanged.

The effect of the new process is clear from the following examples ofthe production of highly immunifying serum.

Example 1 A mule, which, after being treated with the hitherto customaryimmunity method with living red murrain bacillus, when it only gave aserum containing 100 units of immunity, was inoculated with a 150 com.vaccine according to the new process, and four days later again with 150com. of the same vaccine. The value of the serum, after seven more days,had risen to 200 units of immunity and is now so high that the animalwhich was at first put aside as useless could continue to be used forserum production.

Example 2 A horse was to be discarded as useless, as it had not producedserum of high immunity since April 1926, the value of the serum havingsunk below 100 units. It was first inoculated with 150 ccm. vaccineproduced by the new process and the treatment was repeated with 200 cc.of the same vaccine after four days, with another 200 cc. after sevenmore days and with 300 cc. after another three days. The value of itsserum, after twenty-one days from the first inoculation, had risen to150 units of immunity.

It is thus shown that it is possible to immunify animals to a highdegree against the red murrain bacillus by using red murrain bacilliincorporated with aniline dyes. For these experiments an inoculationvaccine completely killed by methyl blue was used, and it was found thatsmall test animals, such as white mice, remained healthy after theinoculation, and the bacilli thus treated could not growv in artificialsoil.

Example 3 The disease affecting poultry known as gapes is an example ofthose diseases, the germs of which have not been isolated up to thepresent. This germ is contained in those organs of the fowl affected bythe disease. Organic extracts are now mixed with a 5% solution of methylblue. With this solution eight fowls were sprayed under the skin eachwith three cc. After fifteen days, these fowls were, together with twountreated test fowl, infected under the skin with 1 cc. organic extract.The untreated test fowl died within five days, while the previouslyinoculated fowl continued to live.

Example 4 It has previously been impossible to immunify birds againstbirds cholera with any vaccine inoculation in such a way that, after asubsequent artificial infection (by means of an injection under the skinor between the muscles), they continued to live. The test birds diedalmost without exception. With birds cholera bacilli killed by fuchsinein the manner above described, one has succeeded, however, inimmunifying birds safely. In this connection, six fowl were inoculatedwith 4 cc. of the new birds cholera vaccine and these fowl, togetherwith a fowl not previously treated, were infected under the skin with0.3 cc. of a 24-hours bouillon culture of birds cholera. The fowl notpreviously treated died of birds cholera after 48 hours, as wasascertained by pathological, anatomical and microscopical examination,while the six fowl previously inoculated remainedalive.

Example 5 The blood serum of a plague peculiar to swine was mixed with1%, 3% and 5% of a 20% brilliant green solution and, after being shakenup, was placed in a hatching-cupboard at a temperature of 3'7 Cent. andleft there for 48 hours. Then three pigs were inoculated, each with 10cc. of these fluids. The three pigs remained healthy, which is evidenceof the fact that the swine plague virus was killed by the addition ofpigment.

After 21 days, the three pigs and another previously untreated test pigwere infected between the muscles with 5 cc. of swine plague virus. Thethree inoculated pigs ate badly on the 7th and 8th days, after theinfection, but otherwise remained healthy, while the test pigs, after afour days illness, died eleven days after infection from the swineplague.

From these examples it is clear that such infectious diseases as arecaused by an unknown germ with a so-called filterable virus againstwhich no effective immunifying agent previously found can likewise becompared, if not stamped out.

Having now described my invention, what I claim and desire to secure byLetters Patent is 1. Process for the manufacture of inoculation vaccinesconsisting in mixing the infectious matter with watery solutions ofaniline dyes and then maintaining the mixture at a blood temperature of37 C. for a period of at least 24 hours up to two days, whereby allbacteria in the infectious matter is killed.

2. In a process according to claim 1, inoculating animals with themixture thus obtained for the purpose of obtaining a serum.

1 WERNER WEICHLEIN.

